A versatile transgenic allele for mouse overexpression studies

For the analysis of gene function in vivo, gene overexpression in the mouse provides an alternative to loss-of-function knock-out approaches and can help reveal phenotypes where compensatory mechanisms are at play. Furthermore, when multiple lines overexpressing a gene-of-interest at varying levels are studied, the consequences of differences in gene dosage can be explored. Despite these advantages, inherent shortcomings in the methodologies used for the generation of gain-of-function transgenic mouse models have limited their application to functional gene analysis, and the necessity for multiple lines comes at a significant animal and financial cost. The targeting of transgenic overexpression constructs at single copy into neutral genomic loci is the preferred method for the generation of such models, which avoids the unpredictable outcomes associated with conventional random integration. However, despite the increased reliability that targeted transgenic methodologies provide, only one expression level results, as defined by the promoter used. Here, we report a new versatile overexpression allele, the promoter-switch allele, which couples PhiC31 integrase-targeted transgenesis with Flp recombinase promoter switching and Cre recombinase activation. These recombination switches allow the conversion of different overexpression alleles, combining the advantages of transgenic targeting with tunable transgene expression. With this approach, phenotype severity can be correlated with transgene expression in a single mouse model, providing a cost-effective solution amenable to systematic gain-of-function studies.

     

Purification of carboxypeptidase B by zinc chelate chromatography

A method is described to purify pancreatic carboxypeptidases B (CPB), removing contaminating endoproteinases that interfere with use of CPB for carboxy-terminal analysis or modification of proteins. The separation uses zinc chelate chromatography and is based on the property that CPB has higher affinity for immobilized zinc ions than do serine proteinases such as trypsin and chymotrypsin, which are abundant endoproteolytic activities in pancreas. CPB preparations are loaded onto a column of iminodiacetic acid-Sepharose that has been saturated with ZnCl2. A step gradient with buffers of decreasing pH is used to elute bound proteins. CPB elutes at a lower pH than do the serine proteinases.     

Sulfation of a gamma-chain variant of human fibrinogen

Biosynthetic sulfation of human fibrinogen was investigated using a hepatoma-derived cell line in culture. Very little [35]sulfate was incorporated into the major forms of the A alpha, B beta, or gamma-chains of fibrinogen, but there was a labeled peptide chain with electrophoretic mobility intermediate between the B beta and gamma-chains. Base hydrolysis of the sulfate-labeled product released tyrosine sulfate. The labeled peptide is identified as a form of gamma-chain by its resistance to proteolysis during extended periods of incubation, in contrast with A alpha and B beta-chains which are converted to smaller forms. The results indicate that human fibrinogen contains tyrosine sulfate primarily within a variant form of the gamma-chain.     

Sulphation of proteins secreted by a human hepatoma-derived cell line. Sulphation of N-linked oligosaccharides on alpha 2HS-glycoprotein.

Several human glycoproteins, including alpha 1-antitrypsin, alpha 1-acid glycoprotein, transferrin, caeruloplasmin and alpha 2HS-glycoprotein, synthesized by the hepatoma-derived cell line HepG2 were observed to contain covalently linked sulphate. These proteins were estimated to contain about 0.1 mol of sulphate/mol of protein. The most abundant of the sulphated glycoproteins, alpha 2HS-glycoprotein, was analysed in detail. All of the sulphate on this protein was attached to N-linked oligosaccharides which contained sialic acid and resisted release by endoglycosidase H. Several independent analytical approaches established that approx. 10% of the molecules of alpha 2HS-glycoprotein contained sulphate. Our results suggest that a number of human plasma proteins contain small amounts of sulphate linked to oligosaccharides.     

Inflammatory protein profile during systemic high dose interleukin-2 administration

Systemic interleukin-2 (IL-2) administration induces an assortment of downstream effects whose biological and therapeutic significance remains unexplored mostly because of the methodological inability to globally address their complexity. Protein array analysis of sera from patients with renal cell carcinoma obtained prior and during high-dose IL-2 therapy had previously revealed extensive alterations in expression of the soluble factors analyzed, whose discovery was limited by the number of capture antibodies selected for protein detection. Here, we expanded the analysis to SELDI-TOF-MS and quantitative protein analysis (nephelometry). All cytokines/chemokines detected by protein arrays were below the SELDI detection limit, while novel IL-2-specific changes in expression of acute-phase reactants and high-density lipoprotein metabolites could be identified. Serum amyloid protein A (SAA) and C-reactive protein expression were consistently up-regulated after four doses of IL-2, while other proteins were down-regulated. These findings were confirmed by SELDI immunoaffinity capture and nephelometry. Immunoaffinity capture revealed different, otherwise undetectable, isoforms of SAA. A linear correlation between peak area by SELDI and protein concentration by nephelometry was observed. Overall distinct yet complementary information was obtained using different platforms, which may better illustrate complex phenomena such as the systemic response to biological response modifiers.     

Quantitative comparison of ligament formulation and pre-strain in finite element analysis of the human lumbar spine

Data has been published that quantifies the nonlinear, anisotropic material behaviour and pre-strain behaviour of the anterior longitudinal, supraspinous (SSL), and interspinous ligaments of the human lumbar spine. Additionally, data has been published on localized material properties of the SSL. These results have been incrementally incorporated into a previously validated finite element model of the human lumbar spine. Results suggest that the effects of increased ligament model fidelity on bone strain energy were moderate and the effects on disc pressure were slight, and do not justify a change in modelling strategy for most clinical applications. There were significant effects on the ligament stresses of the ligaments that were directly modified, suggesting that these phenomena should be included in FE models where ligament stresses are the desired metric.     

Effects of acidotropic compounds on the secretory pathway: inhibition of secretion and processing of the third and fourth components of complement

Acidotropic compounds (also termed lysosomotropic) such as chloroquine and amantadine interfered with processing of the single-chain precursors to the third and fourth components of complement (C3 and C4) by the human hepatoma-derived cell line HepG2. When these compounds were added to culture medium, the precursors of C3 and C4 became the major secretory forms in contrast to the normal secretion of C3 and C4 as their mature forms. In addition, secretion of C3, C4, and total protein was inhibited by these compounds. Our results indicate that lysosomotropic agents, in addition to their well recognized effects on lysosomes and endosomes, inhibit functions of the secretory pathway.     

Alpha 2-antiplasmin's carboxy-terminal lysine residue is a major site of interaction with plasmin

alpha 2-Antiplasmin (AP) inhibits plasmin in a two-step reaction in which AP reversibly binds to lysine-binding sites of plasmin and, then, more slowly complexes covalently with the enzyme's active site. Here, we show that the C-terminal lysine residue of AP has a key role in binding of the inhibitor to plasmin. A synthetic peptide corresponding to the C-terminal 26 amino acid residues of AP blocked association of AP with plasmin, but this activity of the peptide was lost when its C-terminal lysine residue was removed with carboxypeptidase B. The essential role of this lysine residue was shown more directly by treating AP with carboxypeptidase B and observing that AP lost its ability to inhibit plasmin rapidly.